Molecular epidemiology and hematological profiles of hemoglobin variants in southern Thailand

Data on hemoglobin (Hb) variants in southern Thailand are lacking. This study aimed to reassess the frequency of Hb variants and the clinical aspects of compound heterozygous Hb variant with other hemoglobinopathies. We enrolled 13,391 participants from ten provinces in southern Thailand during 2015–2022. Hb analysis was performed using capillary electrophoresis, and mutations in the HBA and HBB genes were identified using PCR or DNA sequencing. Hb variants were identified in 337 (2.5%) unrelated subjects. Nine β-chain variants, namely Hb Malay (76.9%), Hb C (10.1%), Hb D-Punjab (2.9%), Hb G-Makassar (2.3%), Hb Dhonburi (2.3%), Hb Tak (1.4%), Hb J-Bangkok (1.4%), Hb New York (0.3%), and Hb Hope (0.3%), and four α-chain variants—Hb G-Georgia (HBA1) (0.9%), Hb G-Georgia (HBA2) (0.3%), Hb Q-Thailand (0.6%), and Hb St. Luke’s-Thailand (0.3%)—were identified. The southern population exhibited a distinct spectrum of Hb variants compared to that observed in the populations from other areas. Several compound heterozygous genotypes were also identified. Combining Hb Malay with Hb E or high Hb F determinants did not require a blood transfusion. This study provides essential information for genetic counseling in thalassemia prevention and control programs in this region.

Table 5 displays a comparative analysis of the spectrum of Hb variants in the population of southern Thailand and the populations from other areas.The findings indicate distinct prevalence patterns of Hb variants across various parts of the country.Hb Malay and Hb C were the predominant variants in the southern population, whereas Hb Hope, Hb Q-Thailand, and Hb J-Bangkok were frequently found in populations from the northern and central regions.The northeastern population presented the prevalence of four common mutations-Hb

Discussion
This study revisited the molecular spectrum of Hb variants in the southern population across ten provinces through a large-scale survey of specimens received by our center over eight years.Among 13,391 individuals, 337 (2.5%) carried Hb variants, with 263 (78%) identified as Hb variant carriers.The predominant Hb variant was Hb Malay, followed by Hb C. Herein, we report rare Hb variants identified in zone 7, similar to Hb F, including Hb Q-Thailand, Hb Tak, and Hb G-Georgia.To our knowledge, this study is the first to report Hb G-Georgia on the HBA1 gene in Thailand.Hb G-Georgia (HBA1) heterozygote showed lower levels (10.3-10.6%)than  9 .Interestingly, Hb G-Georgia did not present any Hb A 2 variant peak in hemoglobin analysis using the CE technique, observed in both heterozygote Hb G-Georgia and double heterozygote Hb G-Georgia and Hb E. Thus, this could be misconstrued as β + /β + or β + /β 0 -thalassemia disease or β + -thalassemia with Hb E disease based on the Hb pattern, reflecting A 2 FA or EFA.However, the three patients with heterozygous Hb G-Georgia in our study exhibited no clinical symptoms and normal RDW levels.Furthermore, an alkaline denaturation test yielded negative results.Therefore, we performed DNA sequencing to identify this Hb variant.A patient harboring compound heterozygous Hb G-Georgia with α 0 -thalassemia did not develop Hb H disease 4 , indicating that Hb G-Georgia is not classified as an α-thalassemia mutation.However, rapid molecular diagnosis is required for proper genetic counseling.Thus, we developed an allele-specific PCR (AS-PCR) for detecting Hb G-Georgia in both HBA1 and HBA2 genes for the first time (Fig. 4).Unlike PCR-RFLP, this technique is simple, rapid, inexpensive, and does not require restriction enzymes 4 .
Thalassemia mutations are common and heterogeneous in southern populations 13 .We reported the interaction of Hb Malay with other abnormalities resulted in diverse genotypes in 57 (16.9%) patients.The most common genotype was compound heterozygous Hb Malay with Hb E patients (n = 27), manifesting a thalassemia intermedia phenotype without blood transfusion, similar to those reported previously 14,15 .Accordingly, prenatal diagnosis is deemed unnecessary for couples at risk of developing Hb Malay with Hb E disease to reduce the risk of miscarriage.Nevertheless, postnatal diagnosis and appropriate genetic counseling are imperative.This study showed that Hb Malay with β 0 -thalassemia, including codon 17 (A>T), codon 41 (− C), codons 41/42 (− TTCT), IVS1-1 (G>T), or β + -thalassemia, including IVS1-5 (G>C) and IVS2-654 (C>T), led to severe anemia, wherein patients required regular blood transfusion.Prenatal diagnosis is thus essential for families with this combination.Conversely, a patient with Hb Malay and β 0 -thalassemia (3.5 kb deletion) presented with moderate anemia (Hb 9.1 g/dL) without the need for blood transfusion.This milder clinical manifestation could be due to co-inheritance with heterozygous α 0 -thalassemia, ameliorating clinical severity by balancing the levels of α-and β-globin chains [16][17][18][19] .Accordingly, this study supports a previous recommendation proposing the inclusion of α 0 -thalassemia analysis in prenatal diagnosis for fetuses affected with thalassemia disease to make appropriate decisions 20 .Previous studies reported that β 0 -thalassemia (3.5 kb deletion) carriers usually exhibit higher Hb A 2 and Hb F levels than other β-thalassemia carriers due to point mutations 21,22 .The positive result in reverse dot blot (RDB) hybridization indicating a homozygous Hb Malay genotype in the Hb Malay with β 0 -thalassemia (3.5 kb deletion) case, alongside hematological profiles resembling thalassemia intermedia, raises the possibility of misdiagnosis as homozygous Hb Malay.However, the elevated Hb A 2 levels (8.7%) compared to those of homozygous Hb Malay (4.5-5.4%)underscored the need for further laboratory investigation into β 0 -thalassemia (3.5 kb deletion).Subsequently, the true genotype of this patient was found to be Hb Malay with β 0 -thalassemia (3.5 kb deletion).
Interestingly, a previous study reported that Hb Malay with β + -thalassemia typically manifests as a thalassemia intermedia phenotype without the need for regular blood transfusion 15 .However, this study reported three cases of Hb Malay with β + -thalassemia (NT-28 (A>G)) exhibiting distinct phenotypes.One patient presented transfusion-dependent thalassemia and splenomegaly; this was potentially influenced by additional abnormalities.Subsequently, gap-PCR was conducted to identify α-globin gene triplication (ααα/αα) 23 , which, if present, could exacerbate globin chain imbalance and escalate clinical severity 24 .Despite obtaining a negative result for this patient (data not shown), we propose the application of whole-exome sequencing to comprehensively determine the clinical severity.
In Thailand, the frequency of high Hb F determinants is 1.06% 25 .The co-occurrence of this abnormality with β-thalassemia can yield diverse clinical phenotypes, ranging from mild to severe anemia, depending on the β-thalassemia genotype [25][26][27] .However, scant information exists on the clinical phenotypes associated with high Hb F determinants in patients with Hb Malay.We present, for the first time, a case of Hb Malay with Thai  www.nature.com/scientificreports/del-inv-ins A γδβ 0 -thalassemia exhibiting no clinical symptoms.Moreover, combinations of Hb Malay with δβ 0thalassemia (12.5 kb deletion), Indian del-inv A γδβ 0 -thalassemia, or HPFH6 also presented only thalassemia intermedia phenotype without the need for blood transfusion.These results suggest that prenatal diagnosis might be unnecessary for couples carrying Hb Malay with high Hb F determinants.However, a postnatal diagnosis should be performed for proper genetic counseling.Hb J-Bangkok is a β-chain variant occasionally reported in Thailand.A carrier usually presents with normal hematological parameters, with Hb J-Bangkok levels of 44.5 ± 4.7% 28 .However, we report a case of Hb J-Bangkok carrier with moderate anemia (Hb 7.7 g/dL), potentially affected by an underlying disease but unconfirmed patient-specific condition.Interestingly, we report two cases of Hb J-Bangkok with β + -thalassemia (IVS1-5 (G>C) for the first time.Elevated Hb J-Bangkok levels of 89.5% and 93% in patients with mild anemia (Hb 10.1 and 10.6 g/dL) might suggest the presence of homozygous Hb J-Bangkok.However, this rare variant is infrequently reported in southern populations.Thus, these patients are preferably linked to co-inheritance with β-thalassemia mutation, and molecular diagnosis of β-thalassemia is subsequently performed in these cases.
The levels of Hb E or Hb C in pure Hb E or pure Hb C heterozygotes were higher than those genotypes co-inherited with α 0 -thalassemia because the α-globin chain prefers to form dimerization with β-globin chain than β E or β C -globin chain.Thus, reduced α-globin chain production in α 0 -thalassemia contributes to lower Hb E or Hb C levels.However, Hb E levels in compound heterozygous Hb C/Hb E are higher than in Hb E heterozygote.The previous studies reported that the Hb E levels in compound heterozygous Hb C/Hb E could be presented in a wide range from 32.0 to 39.7% 14,29 .This study reported four cases with compound heterozygous Hb C/Hb E without co-inherited α-thalassemia with Hb E levels ranging from 30.6 to 34.0%, while a compound heterozygous Hb C/Hb E co-inherited α 0 -thalassemia presented with Hb E levels of 35.6%.Hence, lower levels of Hb E were not observed in a compound heterozygous Hb C/Hb E co-inherited with α 0 -thalassemia.It might be due to both Hb E and Hb C are positively charged Hb variants, which might have a similar ability to interact with the α-globin chain.Moreover, the decrease of αβ C dimer formation leading to an indirect increase in the αβ E dimer formation 29 as the same as that presented in compound heterozygous Hb S/Hb E disease 30 .
For two cases of compound heterozygous Hb D-Punjab/Hb E co-inherited with α + -thalassemia, the Hb D-Punjab value (66.6% and 65.6%) is elevated while the Hb E value (24.5% and 29.0%) is the same as the Hb E heterozygote.Hb D-Punjab mutation results in structural protein changes but does not affect the value of Hb D-Punjab production.However, Hb E mutation creates abnormal mRNA splicing, resulting in low output of Hb E. Thus, lower Hb E levels than Hb D-Punjab levels could be observed in the compound heterozygous Hb D-Punjab/Hb E cases.Moreover, co-inherited α + -thalassemia in compound heterozygous Hb D-Punjab/Hb E cases might not much affect the lower production of Hb E levels when compared to Hb E levels of compound heterozygous Hb D-Punjab/Hb E with normal α-globin chain cases in a previous report (24.5% and 29.0% vs 28.4% and 29.3%) 31 .in West African populations 37 and is occasionally reported in Southeast Asian populations of different origins 29 .Carriers of Hb C are immune to malarial infections 38 .Accordingly, Hb C is predominantly observed in southern Thailand, where malaria is endemic.Moreover, Hb G-Makassar is frequently observed in the southern population, similar to that in the Malaysian population 39 .Hb G-Makassar comigrated at the same retention time as that for Hb S, as determined using the CE technique.Thus, molecular testing is essential for differential diagnosis.Furthermore, we reported two cases of Hb G-Makassar with Hb E, presenting mild clinical phenotypes similar to that in a previous report 39 .
In conclusion, this study demonstrates a distinct spectrum of Hb variants in Thailand.In addition, we describe the clinical aspects of Hb variants in combination with thalassemia or hemoglobinopathies.This information is essential for determining the need to perform prenatal diagnosis in the prevention and control program for thalassemia in this region.

F ( 11 .
6%) levels.In contrast, a patient with compound heterozygous Hb C and β 0 -thalassemia (codon 41 (− C)) exhibited Hb A 2 (3.7%) without detectable Hb F levels.This study reports several Hb variants co-inherited with Hb E in the southern population, including two cases of Hb D-Punjab/Hb E, two cases of Hb G-Makassar/Hb E, one case of Hb J-Bangkok/Hb E, and one case of Hb Hope/Hb E, all of which showed no clinical symptoms.

Figure 1 .
Figure 1.Distribution of Hb variants among 346 chromosomes of 337 participants across ten provinces of southern Thailand.The number represents the allelic count of Hb variants.

Figure 3 .
Figure 3. Hemoglobin (Hb) analysis results of Hb variants from southern populations using the capillary electrophoresis method.

Table 1 .
Mutation frequencies of hemoglobin (Hb) variants in a total of 346 chromosomes among 337 participants from each province of southern Thailand during 2015 to 2022.The number represents the allelic count of Hb variants.

Table 2 .
Hematological characteristics of 263 Hb variant carriers.a Hb variant co-migrated with Hb A, b Hb variant co-migrated with Hb F, NA not available, Hb Var hemoglobin variant, CS Constant Spring, F female, M male, CD codon.

Table 3 .
Hematological characteristics of 61 patients with homozygous or compound heterozygous Hb variants and hemoglobinopathies who were not dependent on transfusion.a Hb variant co-migrated with Hb F, NA not available, Hb Var hemoglobin variant, thal thalassemia, F female, M male, NT nucleotide, IVS intervening sequence.

Table 4 .
Hematological characteristics of 13 patients with compound heterozygous Hb variants and hemoglobinopathies who were dependent on transfusion.NA not available, thal thalassemia, F female, M male, NT nucleotide, IVS intervening sequence.